Novel approaches to prevent the acquisition of human immunodeficiency virus type 1 (HIV) by vaccination or pre-exposure prophylaxis are being vigorously pursued, but for the more than 34 million people currently living with HIV, it remains an incurable disease that can only be treated with lifelong daily antiretroviral therapy. As such, a cure for HIV remains a high priority. The indefinite persistence of latent, replication-competent HIV in long-lived CD4+ T cells is a major obstacle to achieving a cure. Innovative therapies are being developed to target this latent reservoir, but simpler, higher throughput and more precise measures are needed to accelerate progress. In this proposal, we outline a research strategy that will evaluate innovative cell culture and molecular assays of the latent reservoir of HIV that have the potential for higher throughput, greater precision and lower cost than the current gold standard assay of infectious virus recovery (IVR). The three specific aims of this proposal are: 1) to further develop and assess a simplified, high-throughput, precise and lower cost cell culture-based assay of total inducible virus recovery (TVR) from blood-derived resting CD4+ T cells (rCD4 cells) that can be performed on either fresh or cryopreserved peripheral blood mononuclear cells (PBMC); 2) to compare, using rCD4 cells isolated from the same donor, the performance characteristics of the optimized TVR assay developed in Aim 1 to that of a simplified IVR assay that uses an HIV susceptible cell line (Molt-4) rather than allogeneic blasts as target cells; and 3) to evaluate the relationships between TVR and IVR assay results and molecular measures of HIV persistence, including enhanced qPCR assays of residual plasma viremia, cellular HIV RNA species, and HIV RNA to DNA ratios in rCD4 cells to identify a reliable molecular surrogate of the latent but inducible HIV reservoir. Completion of Aims 1-3 should provide urgently needed tools to measure changes in the latent HIV reservoir following therapeutic interventions. The proposed work will develop and evaluate both cell culture (inducible virus recovery) and molecular assays of the latent reservoir. As such, the goals of this project are closely aligned with RFA-AI-13-038. The availability of simplified assays to quantify latent HIV will undoubtedly accelerate progress towards a cure by facilitating proof-of-concept studies of innovative therapies.